Methods and devices of the kind under discussion here have long been known in actual practice and they are used especially in fluorescence microscopy. In the realm of fluorescence microscopy, a specimen to be examined is often dyed with several fluorescent dyes so that the aspect of separating the individual emission wavelengths is of paramount significance. With the methods known so far, the specimen is first illuminated for a certain length of time with a first excitation wavelength and data is then gathered in order to be able to generate a first image of the specimen in this manner. Subsequently, the specimen is illuminated for the same length of time with a second excitation wavelength and thus a second image of the specimen is obtained. This process is carried out consecutively for all excitation wavelengths of the fluorescent dyes contained in the specimen, so as to obtain a discrete image for each excitation wavelength. The process can be performed linewise as well as imagewise, which is known by the designation “sequential scan”.
A particular drawback associated with the prior-art sequential scan is that the specimen has to be scanned several times in order to generate the individual images. This procedure is extremely time-consuming since the imaging speed is essentially limited by the speed of the scanning procedure. Thus, in comparison to normal imaging, approximately twice as much time is needed to create separate images for two excitation wavelengths and about three times as much time is needed for three excitation wavelengths. As a result, the imaging rate in the confocal scanning microscope is significantly reduced.
Moreover, the sequential scan entails problems when the image changes rapidly, for instance, due to processes transpiring inside the specimen over a short period of time. When such rapid processes take place, differences appear in the individual images since the images for different excitation wavelengths are not taken simultaneously. This is particularly critical with the bidirectional linewise sequential scan, since in this case, there is not even a fixed time relationship between the individual pixels. When the image data thus obtained is further processed with conventional methods, for example, by means of spectrum deconvolution or linear unmixing, artifacts show up in the processed images.